Polypeptides having lysozyme activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having lysozyme activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/495,031filed on Apr. 24, 2017, now U.S. Pat. No. 10,039,300, which is adivisional of U.S. Ser. No. 14/360,508 filed on May 23, 2014, now U.S.Pat. No. 9,663,775, which is a 35 U.S.C. 371 national application ofinternational application no. PCT/EP2012/073493 filed Nov. 23, 2012,which claims priority or the benefit under 35 U.S.C. 119 of Europeanapplication no. 11190690.5 filed Nov. 25, 2011 and U.S. provisionalapplication No. 61/564,372 filed Nov. 29, 2011. The content of eachapplication is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having lysozyme activity,catalytic domains, and polynucleotides encoding the polypeptides andcatalytic domains. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides and catalyticdomains.

Description of the Related Art

Lysozyme is an O-glycosyl hydrolase produced as a defensive mechanismagainst bacteria by many organisms. The enzyme causes the hydrolysis ofbacterial cell walls by cleaving the glycosidic bonds of peptidoglycan;an important structural molecule in bacteria. After having their cellwalls weakened by lysozyme action, bacterial cells lyse resulting fromosmotic pressure.

Lysozyme occurs in many organisms such as viruses, plants, insects,birds, reptiles and mammals. In mammals, Lysozyme has been isolated fromnasal secretions, saliva, tears, intestines, urine and milk. The enzymecleaves the glycosidic bond between carbon number 1 of N-acetylmuramicacid and carbon number 4 of N-acetyl-D-glucosamine. In vivo, these twocarbohydrates are polymerized to form the cell wall polysaccharide.

There is an increasing interest in the potential of lysozyme enzymes asantimicrobial agents. For example, lysozyme activity has been shownagainst pathogens such as Streptococcus pneumoniae, Bacillus anthracis,Enterococcus faecium, Bacillus stearothermophilus, Clostridiumbotulinum, Clostridium butyricum, Clostridium perfringens, Clostridiumsporogenes, Clostridium tyrobutyricum, and Listeria monocytogenes.

Lysozyme has been classified into five different glycoside hydrolase(GH) families (CAZy, www.cazy.org): hen egg-white lysozyme (GH22), gooseegg-white lysozyme (GH23), bacteriophage T4 lysozyme (GH24),Sphingomonas flagellar protein (GH73) and Chalaropsis lysozymes (GH25).Lysozymes from the families GH23 and GH24 are primarily known frombacteriophages and have not been identified in fungi. The lysozymefamily GH25 has been found to be structurally unrelated to the otherlysozyme families.

Use of lysozyme has been suggested in animal feed (see for example WO00/21381 and WO 2004/026334), in cheese production (see for example WO2005/080559), food preservation (Hughey and Johnson, 1987, Appl.Environ. Microbiol. 53:2165), detergents (see for example U.S. Pat. No.5,041,236 and EP 0425016), in oral care (see for example U.S. Pat. No.4,355,022, WO 2004/017988 and WO 2008/124764), cosmetology anddermatology, contraception, urology, and gynaecology (see for example WO2008/124764).

A GH25 lysozyme has been reported from Chalaropsis (Felsch et al., 1975,“The N,O-Diacetylmuramidase of Chalaropsis species; V The complete aminoacid sequence”, J. Biol. Chem. 250(10):3713-3720).

Hen egg white lysozyme which is the primary product available on thecommercial market, does not cleave N,6-O-diacetylmuramidase in, e.g.,Staphylococcus aureus cell walls and is thus unable to lyse thisimportant human pathogen among others (Masschalck et al., 2002, “Lyticand nonlytic mechanism of inactivation of gram-positive bacteria bylysozyme under atmospheric and high hydrostatic pressure”, J. Food Prot.65(12):1916-23).

It has been observed that different lysozymes have differentspecificities towards different microorganisms. It is thereforedesirable to have several lysozymes available in order to be able toselect suitable enzymes for each particular application. Newpolypeptides having lysozyme activity is therefore desired.

SUMMARY OF THE INVENTION

The present invention related to isolated fungal polypeptides belongingto the GH23 or GH24 families and having lysozyme activity.

The present invention further relates to isolated polypeptides havinglysozyme activity selected from the group consisting of:

(a) a polypeptide having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 6;

(b) a polypeptide having at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 2;

(c) a polypeptide having at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to themature polypeptide of SEQ ID NO: 4;

(d) a polypeptide encoded by a polynucleotide having at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1;

(e) a polypeptide encoded by a polynucleotide having at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the mature polypeptide coding sequence of SEQID NO: 3;

(f) a polypeptide encoded by a polynucleotide having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 5;

(g) a polypeptide encoded by a polynucleotide that hybridizes undermedium-high stringency conditions with the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or thefull-length complement of thereof;

(h) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 6, comprising a substitution, deletion, and/or insertion atone or more (e.g., several) positions; and

(i) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g)or (h) that has lysozyme activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to the polypeptides on the inventionhaving antimicrobial activity and methods of use of the polypeptides onthe invention as inhibitors of biofilm formation, in detergentcompositions, in animal feed and for the extraction of bacterial genomicDNA.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2,amino acids 1 to 20 of SEQ ID NO: 4 or amino acids 1 to 19 of SEQ ID NO:6, which is operably linked to a gene encoding a protein; nucleic acidconstructs, expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing a protein.

Overview of Sequence Listing

SEQ ID NO: 1 is the DNA sequence of the P8EH GH23 gene as isolated fromAspergillus aculeatus CBS 172.66.

SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1.

SEQ ID NO: 3 is the DNA sequence of the P242MS GH24 gene as isolatedfrom Acremonium alkalophilum CBS 114.92.

SEQ ID NO: 4 is the amino acid sequence as deduced from SEQ ID NO: 3.

SEQ ID NO: 5 is the DNA sequence of the P244A7 GH24 gene as isolatedfrom Acremonium alkalophilum CBS 114.92.

SEQ ID NO: 6 is the amino acid sequence as deduced from SEQ ID NO: 5.

SEQ ID NO: 7 is the DNA sequence of the P242M9 GH25 gene as isolatedfrom Acremonium alkalophilum CBS 114.92.

SEQ ID NO: 8 is the amino acid sequence as deduced from SEQ ID NO: 7.

SEQ ID NO: 9 is the forward primer F-P8EH.

SEQ ID NO: 10 is the reverse primer R-P8EH.

SEQ ID NO: 11 is the forward primer F-P242MS.

SEQ ID NO: 12 is the reverse primer R-P242MS.

SEQ ID NO: 13 is the forward primer F-P244A7.

SEQ ID NO: 14 is the reverse primer R-P244A7.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows radial diffusion assays of Acremonium alcalophilum GH24lysozyme (EXP03890, SEQ ID NO: 6), Acremonium alcalophilum GH25 lysozyme(EXP03864, SEQ ID NO: 8) and a reference lysozyme from Aspergillusfumigatus GH25 on S. carnosus and E. coli.

DEFINITIONS

Lysozyme: The term “lysozyme” activity is defined herein as anO-glycosyl hydrolase, which catalyses the hydrolysis of the glycosidicbond between two or more carbohydrates, or between a carbohydrate and anon-carbohydrate moiety. Lysozymes cleave the glycosidic bond betweencertain residues in mucopolysaccharides and mucopeptides of bacterialcell walls, such as 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins, resulting inbacteriolysis. Lysozyme belongs to the enzyme class EC 3.2.1.17. Forpurposes of the present invention, lysozyme activity is determinedaccording to the turbidity assay described in example 5. In one aspect,the polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the lysozyme activity ofthe mature polypeptide of one of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ IDNO: 6.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Antimicrobial activity: The term “antimicrobial activity” is definedherein as is an activity that kills or inhibits the growth ofmicroorganisms, such as, algae, archea, bacteria, fungi and/orprotozoans. The antimicrobial activity can for example be bactericidalmeaning the killing of bacteria or bacteriostatic meaning the preventionof bacterial growth. The antimicrobial activity can include catalyzingthe hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins. Antimicrobial activitycan also include the lysozyme binding to the surface of themicroorganism and inhibiting its growth. The antimicrobial effect canalso include the use of the lysozymes of the present invention foractivation of bacterial autolysins, as an immunostimulator, byinhibiting or reducing bacterial toxins and by an opsonin effect. Forpurposes of the present invention, antimicrobial activity is determinedaccording to the radial diffusion assay described in example 4.

Altered/modified property: The term “altered/modified property” isdefined herein as a characteristic associated with a variant that isaltered or modified, as compared relative to the parent lysozyme or anidentified reference sequence. The altered or modified property may be acharacteristic associated with a variant that is improved, unlessotherwise stated, relative to another reference lysozyme or the parentlysozyme. Examples of properties which can be altered/modified orimproved are given below.

Thermostability: The term “thermostability” refers to the lysozymeactivity after a period of incubation at elevated temperature relativeto the parent or an identified reference sequence, either in a buffer orunder conditions such as those which exist during productstorage/transport or conditions similar to those that exist duringindustrial use of the variant. A variant may or may not display analtered thermal activity profile relative to the parent. In one aspect,the thermostability of the variant having lysozyme activity is at least1.0-fold, e.g., at least 1.1-fold, at least 1.5-fold, at least 1.8-fold,at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, atleast 20-fold, and at least 25-fold more thermostable than the parent orreference sequence at the selected temperature. Preferably the activityis tested using the lysozyme turbidity activity assay described in the“Materials and Methods” section.

Temperature profile/temperature stability: The term “temperatureprofile/temperature stability” refers to the variant enzyme showing amodified temperature profile as compared to the parent or an identifiedreference sequence, wherein the temperature profile is determined aslysozyme activity as a function of temperature. The activity at eachtemperature is preferably indicated as relative activity (in %)normalized to the value at optimum temperature. The optimum temperatureis that temperature within the tested temperatures (i.e., those with5-10° C. jumps) where the activity is highest.

pH stability: The term “pH stability” refers to the variant enzymedisplaying structural stability relative to the parent lysozyme or anidentified reference sequence, after a period of incubation at a pHwhich is outside the pH range where the enzyme is active (pH activityrange). Such a variant may or may not display an altered pH activityprofile relative to the parent. For example, the variant may not beactive at the increased or decreased pH, but is able to maintain itsthree dimensional structure and then regain activity once it is returnedto the pH activity range. Alternatively, the variant may have animproved ability to refold relative to the parent following incubationat increased or decreased pH.

In one aspect, the pH stability profile is altered such that a lysozymevariant has improved stability at acidic pH. As used herein, acidic pHmeans from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferablyfrom 3 to 5, even more preferably from 3.5 to 4. Preferably, the variantlysozyme maintains at least 40%, preferably at least 50%, 60%, 70% or80%, more preferably at least 90%, even more preferably at least 95%residual activity after incubation at a given pH for 1 hour whencompared to the variant which has been maintained at pH 6.5 for the sametime. Preferably, the residual activity of the variant lysozyme is atleast 1.1-fold, at least 1.3-fold, at least 1.5-fold, preferably atleast 2-fold, more preferably at least 5-fold, most preferably at least7-fold, and even most preferably at least 10-fold higher than theresidual activity of the parent lysozyme or an identified referencesequence which has been treated under the same conditions. Preferably,the activity is tested using the lysozyme turbidity activity assaydescribed in the “Materials and Methods” section.

pH activity profile: The term “pH activity profile” is defined herein asa variant lysozyme displaying an alteration of the pH-dependent activityprofile when compared to the pH activity profile of the parent lysozymeor an identified reference sequence. The pH activity profile provides ameasure of the enzyme's efficiency in preventing microbial growth,eliminating microbial cells and/or performing catalysis of a hydrolysisreaction over a pH range at given conditions such as temperature andsolvent composition. A lysozyme has a specific pH range wherein thepolypeptide is stable and retains its enzymatic activity, outside thisrange the lysozyme becomes less active and potentially also less stable.Within the pH range there generally is a pH optimum, where the lysozymeshows the highest activity.

A lysozyme variant with improved activity at alkaline pH (e.g., from pH7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, evenmore preferably from 9 to 9.5) will be able to function in more alkalineenvironments such as detergents.

A variant with improved activity at acidic pH (e.g., from pH 2 to 6.5,preferably from 2.5 to 6, more preferably from 3 to 5.5, even morepreferably from 3.5 to 5) will be able to function under more acidicconditions, such as preservative in certain foods or as a eubioticmolecule in feeds.

In one aspect, the pH activity profile is altered such that a lysozymevariant has improved activity at a more alkaline pH. Preferably, theactivity of the lysozyme variant at a pH at least 0.5 units higher,preferably at least 1.0 pH units higher, more preferably at least 1.5 pHunits higher, even more preferably at least 2.0 pH units higher is atleast 1.1-fold, preferably at least 1.5-fold, more preferably at least2-fold, even more preferably at least 5-fold and most preferably atleast 10-fold higher than that of the parent enzyme or an identifiedreference sequence. Preferably, the lysozyme variant at the same timemaintains at least 40%, preferably at least 50%, 60%, 70% or 80%, or90%, more preferably at least 95%, even more preferably at least 100% ofthe activity that parent lysozyme or an identified reference sequenceexhibits at its pH optimum. Preferably, the activity is tested using thelysozyme turbidity activity assay described in the “Materials andMethods” section.

In another aspect, the pH activity profile is altered such that alysozyme variant has improved activity at a more acidic pH. Preferably,the activity of the lysozyme variant at a pH at least 0.5 units lower,preferably at least 1.0 pH units lower, more preferably at least 1.5 pHunits lower, even more preferably at least 2.0 pH units lower is atleast 1.1-fold, preferably at least 1.5-fold, more preferably at least2-fold, even more preferably at least 5-fold and most preferably atleast 10-fold higher than that of the parent enzyme or an identifiedreference sequence. Preferably, the lysozyme variant at the same timemaintains at least 40%, preferably at least 50%, 60%, 70% or 80%, or90%, more preferably at least 95%, even more preferably at least 100% ofthe activity that parent lysozyme or an identified reference sequenceexhibits at its pH optimum. Preferably, the activity is tested using thelysozyme turbidity activity assay described in the “Materials andMethods” section.

Glycation Susceptibility: Non-enzymatic glycation is a spontaneousposttranslational process where reducing sugars bind covalently to freeamino groups in proteins primarily at Lysine (K) residues. Glycation mayimpact the activity of the lysozyme. In accordance with the presentinvention, the susceptibility of the lysozyme to non-enzymatic glycationmay be reduced by specified amino acid changes.

Improved properties may also include thermal properties, such aspelleting stability, steam stability, broader temperature activityprofile. Further improved properties may include protease-sensibility,and/or glycosylation pattern. Improvements are preferably assessed inrelation to the desired application conditions.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide or a catalytic domainhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or domain; wherein thefragment has lysozyme activity. In one aspect, a fragment contains atleast 198 amino acid residues (e.g., amino acids 59 to 256 of SEQ ID NO:2), or at least 230 amino acid residues (e.g., amino acids 32 to 261 ofSEQ ID NO: 2). In another aspect, a fragment contains at least 159 aminoacid residues (e.g., amino acids 25 to 183 of SEQ ID NO: 4). In afurther aspect, a fragment contains at least 150 amino acid residues(e.g., amino acids 24 to 173 of SEQ ID NO: 6).

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 20 to 264 of SEQ ID NO: 2, amino acids 21 to186 of SEQ ID NO: 4 or amino acids 20 to 176 of SEQ ID NO: 6 based onthe SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6)that predicts amino acids 1 to 19 of SEQ ID NO: 2, amino acids 1 to 20of SEQ ID NO: 4 and amino acids 1 to 19 of SEQ ID NO: 6 are signalpeptides. It is known in the art that a host cell may produce a mixtureof two of more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lysozyme activity. In one aspect, the mature polypeptide codingsequence is the joint sequence of nucleotides 58 to 571 and nucleotides639 to 859 of SEQ ID NO: 1, the joint sequence of nucleotides 61 to 267and nucleotides 335 to 625 of SEQ ID NO: 3 or the joined sequence ofnucleotides 58 to 133, nucleotides 215 to 345 and nucleotides 516 to 779of SEQ ID NO: 5 based on the SignalP program (Nielsen et al., 1997,supra) that predicts nucleotides 1 to 57 of SEQ ID NO: 1, nucleotides 1to 60 of SEQ ID NO: 3 and nucleotides 1 to 57 of SEQ ID NO: 5 encodesignal peptides.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

Stringency conditions: The different stringency conditions are definedas follows.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lysozyme activity. In one aspect, a subsequence containsat least 594 nucleotides (e.g., the joint sequence of nucleotides 175 to571 and nucleotides 639 to 835 of SEQ ID NO: 1), or at least 690nucleotides (e.g., the joint sequence of nucleotides 94 to 571 andnucleotides 639 to 850 of SEQ ID NO: 1). In another aspect, asubsequence contains at least 477 nucleotides (e.g., the joint sequenceof nucleotides 73 to 267 and nucleotides 335 to 616 of SEQ ID NO: 3). Ina further aspect, a subsequence contains at least 450 nucleotides (e.g.,the joint sequence of nucleotides 70 to 133, nucleotides 215 to 345 andnucleotides 516 to 770 of SEQ ID NO: 5).

Substantially pure polynucleotide: The term “substantially purepolynucleotide” means a polynucleotide preparation free of otherextraneous or unwanted nucleotides and in a form suitable for use withingenetically engineered polypeptide production systems. Thus, asubstantially pure polynucleotide contains at most 10%, at most 8%, atmost 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, andat most 0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. Preferably, thepolynucleotide is at least 90% pure, e.g., at least 92% pure, at least94% pure, at least 95% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99% pure, and at least 99.5% pure by weight.The polynucleotides of the present invention are preferably in asubstantially pure form.

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well known recombinantmethods or by classical purification methods.

Variant: The term “variant” means a polypeptide having lysozyme activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, of one or more (several) amino acid residues at one or more(e.g., several) positions. A substitution means replacement of the aminoacid occupying a position with a different amino acid; a deletion meansremoval of the amino acid occupying a position; and an insertion meansadding 1, 2, or 3 amino acids adjacent to and immediately following theamino acid occupying the position. A variant according to the inventionmay comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from1 to 52, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52alterations.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Lysozyme Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 80% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 85% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 90% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 91% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 92% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 93% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 94% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 95% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 96% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 97% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 98% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 99% which have lysozyme activity.

In one aspect, the polypeptides differ by no more than 52 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51, from themature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having lysozyme activity. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 2. In another aspect, the polypeptide comprises orconsists of amino acids 20 to 264 of SEQ ID NO: 2.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 85% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 90% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 91% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 92% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 93% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 94% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 95% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 96% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 97% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 98% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 4 ofat least 99% which have lysozyme activity.

In one aspect, the polypeptides differ by no more than 27 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25 or 26, from the mature polypeptide of SEQ ID NO:4.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 4 or an allelic variantthereof; or is a fragment thereof having lysozyme activity. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 4. In another aspect, the polypeptide comprises orconsists of amino acids 21 to 186 of SEQ ID NO: 4.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 90% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 91% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 92% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 93% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 94% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 95% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 96% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 97% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 98% which have lysozyme activity.

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 ofat least 99% which have lysozyme activity.

In one aspect, the polypeptides differ by no more than 17 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, from themature polypeptide of SEQ ID NO: 6.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 6 or an allelic variantthereof; or is a fragment thereof having lysozyme activity. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 6. In another aspect, the polypeptide comprises orconsists of amino acids 20 to 176 of SEQ ID NO: 6.

In another embodiment, the present invention relates to an isolatedpolypeptide having lysozyme activity encoded by a polynucleotide thathybridizes under medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with the mature polypeptide coding sequence of (i) SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 5, (ii) the cDNA sequence thereof, or(iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or asubsequence thereof, as well as the polypeptide of SEQ ID NO: 2, SEQ IDNO: 4 or SEQ ID NO: 6 or a fragment thereof, may be used to designnucleic acid probes to identify and clone DNA encoding polypeptideshaving lysozyme activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic DNA or cDNA of a cell ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having lysozyme activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ IDNO: 5 or a subsequence thereof, the carrier material is used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; (ii) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:5; (iii) the cDNA sequence thereof (iv) the full-length complementthereof; or (v) a subsequence thereof; under medium to very highstringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is nucleotides 58 to 571 ornucleotides 639 to 859 of SEQ ID NO: 1, nucleotides 61 to 267 ornucleotides 335 to 625 of SEQ ID NO: 3, or nucleotides 58 to 133,nucleotides 215 to 345 or nucleotides 516 to 779 of SEQ ID NO: 5. Inanother aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; themature polypeptide thereof; or a fragment thereof. In another aspect,the nucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 orthe cDNA sequence thereof.

In another embodiment, the present invention relates to an isolatedpolypeptide having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof of at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the present invention relates to an isolatedpolypeptide having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 3 or the cDNA sequence thereof of at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to an isolatedpolypeptide having lysozyme activity encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5 or the cDNA sequence thereof of at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is notmore than 52, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or51.

In a further embodiment, the present invention relates to variants ofthe mature polypeptide of SEQ ID NO: 4 comprising a substitution,deletion, and/or insertion at one or more (e.g., several) positions. Inan embodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 4 is notmore than 27, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26.

In an additional embodiment, the present invention relates to variantsof the mature polypeptide of SEQ ID NO: 6 comprising a substitution,deletion, and/or insertion at one or more (e.g., several) positions. Inan embodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 6 is notmore than 17, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or16.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lysozyme activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Lysozyme Activity

A polypeptide having lysozyme activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a filamentous fungal polypeptide such as anAcremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Penicillium,Thielavia or Trichoderma polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

In another aspect, the polypeptide is an Aspergillus aculeatus or anAcremonium alcalophilum polypeptide, e.g., a polypeptide obtained fromAspergillus aculeatus CBS 172.66 or Acremonium alcalophilum CBS 114.92.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Aspergillus orAcremonium, or a related organism and thus, for example, may be anallelic or species variant of the polypeptide encoding region of thepolynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5 or the cDNA sequences thereof, e.g., a subsequencethereof, and/or by introduction of nucleotide substitutions that do notresult in a change in the amino acid sequence of the polypeptide, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCI B 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMε1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to19 of SEQ ID NO: 2, amino acids 1 to 20 or SEQ ID NO: 4 or amino acids 1to 19 of SEQ ID NO: 6. The polynucleotides may further comprise a geneencoding a protein, which is operably linked to the signal peptide. Theprotein is preferably foreign to the signal peptide. In one aspect, thepolynucleotide encoding the signal peptide is nucleotides 1 to 57 of SEQID NO: 1, nucleotides 1 to 60 of SEQ ID NO: 3 or nucleotides 1 to 57 ofSEQ ID NO: 5.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will depend to a large extent upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M.I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is an Aspergillus or an Acremonium cell.In a more preferred aspect, the cell is an Aspergillus aculeatus or anAcremonium alcalophilum cell. In a most preferred aspect, the cell isAspergillus aculeatus CBS 172.66 or Acremonium alcalophilum CBS 114.92.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides for example the lysozyme spot assay asdescribed below. These detection methods include, but are not limitedto, use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germ plasma. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Uses

Examples of preferred uses of the lysozyme or compositions thereof ofthe present invention are given below. The dosage of the lysozyme andother conditions under which the lysozyme is used may be determined onthe basis of methods known in the art.

The polypeptides of the invention are typically useful at any locussubject to contamination by bacteria, fungi, yeast or algae. Typically,loci are in aqueous systems such as cooling water systems, laundry rinsewater, oil systems such as cutting oils, lubricants, oil fields and thelike, where it is desired to kill the microorganisms or at least tocontrol their growth. However, the present invention may also be used inall applications for which known lysozymes compositions are useful, suchas protection of wood, latex, adhesive, glue, paper, cardboard, textile,leather, plastics, caulking, and feed.

A lysozyme, or a composition thereof, of the present invention may beused in several applications to degrade a material comprising apeptidoglycan or a chitodextrin by treating the material with thelysozyme or composition thereof (see for example Proctor and Cunningham,1988, Critical Reviews in Food Science and Nutrition 26:359-395; Cariniet al., 1985, Microbiol. Alimen. Nutr. 3:299-320; Hughey and Johnson,1987, Appl. Environ. Microbiol. 53:2165-2170; Cunningham et al., 1991,World's Poultry Science Journal 47:141-163).

Uses of Lysozymes of the Invention for Cleaning and/or Detergents

A lysozyme of the present invention is preferably incorporated intoand/or used together with detergent compositions as described below.When washing is performed repeatedly at temperatures below 60° C. thereis an increased risk of malodour in the washing machine (laundry as wellas dishwashing) and on the textiles or items washed in the machine. Thismalodour is likely to be caused by microbial organisms such as bacteria,fungi, algae or other unicellular organisms growing in the washingmachine.

Furthermore, the invention relates to a process for laundering offabrics comprising treating fabrics with a washing solution containing adetergent composition and a lysozyme or a lysozyme composition of theinvention. The laundering treatment can for example be carried out in amachine washing process or in a manual washing process. The washingsolution can for example be an aqueous washing solution containing thedetergent composition and with a pH between 3 and 12.

The fabrics subjected to the methods of the present invention may beconventional washable laundry, for example household laundry.Preferably, the major part of the laundry is garments and fabrics,including knits, wovens, denims, yarns, and toweling, made from cotton,cotton blends or natural or manmade cellulosics (e.g., originating fromwood pulp) or blends thereof. Examples of blends are blends of cotton orrayon/viscose with one or more companion material such as wool,synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g., rayon/viscose, ramie, flax/linen,jute, cellulose acetate fibers, lyocell).

The present invention provides a method of reducing microbialcontamination on a surface, such as a textile garment or hard surfacesuch as metal, plastic or rubber parts in a washing machine or dishwashing machine, bathroom tiles, floors, table tops, drains, sinks andwashbasin, by treating the microbially contaminated surface with alysozyme or lysozyme composition of the present invention. Such atreatment is also expected to reduce the malodour on textiles and hardsurfaces containing microbial contamination.

The reduction of microbial contamination can be assessed in severalways, for example by letting a panel assess whether the smell has beendecreased, alternatively a sample may be taken from the surface andcultivated to assess whether the microbial count has been reduced as aresult of the treatment compared to a treatment without lysozyme.

Uses of Lysozymes of the Invention in Animal Feed

A lysozyme of the invention may also be used in animal feed. In anembodiment, the present invention provides a method for preparing ananimal feed composition comprising adding a lysozyme of the presentinvention to one or more animal feed ingredients.

A lysozyme of the present invention may for example be used to stabilizethe healthy microflora of animals, in particular livestock such as, butnot limited to, sheep, goats, cattle (including, but not limited to,beef cattle, cows, and young calves), deer, pigs or swine (including,but not limited to, piglets, growing pigs, and sows), poultry(including, but not limited to, geese, turkeys, ducks and chicken suchas broilers, chicks and layers); horses, moose and rabbits but also infish (including but not limited to salmon, trout, tilapia, catfish andcarps; and crustaceans (including but not limited to shrimps andprawns)). In a preferred embodiment, the lysozyme replaces antibioticsin animal diets. In a further embodiment, a lysozyme of the presentinvention is used as a feed additive, where it may provide a positiveeffect on the microbial balance of the chicken digestive tract and inthis way improve animal performance.

A lysozyme of the present invention may also be used in animal feed asfeed enhancing enzymes that improve feed digestibility to increase theefficiency of its utilization according to WO 00/21381 and WO2004/026334.

In a further embodiment, a lysozyme of the present invention may be usedas a feed additive, where it may provide a positive effect on theanimals digestive tract and in this way improve animal performance inaccordance to weight gain, feed conversion ratio (FCR), or improvedanimal health such as decreased mortality rate. FCR is calculated as thefeed intake in g/animal relative to the weight gain in g/animal.

Uses of Lysozymes of the Invention as Antimicrobial Agents

A lysozyme of the present invention may be used as antimicrobial agents.One aspect of the present invention is a method for reducing microbialcontamination, comprising treating a microbial contaminated surface witha lysozyme of the present invention.

To assess whether a lysozyme of the present invention is capable ofacting as an antimicrobial agent it can be tested in a turbidity assay.In this assay it is tested whether the lysozyme is capable of degradingmicrobial cells, e.g., a dried substrate of Exiguobacterium undae cells(isolated from a smelly sock) or Micrococcus luteus cells dissolved inbuffer or detergent, and thereby reducing the optical density (OD) atfor example 540 nm, when compared to a microbial suspension only treatedwith buffer.

Uses of Lysozymes of the Invention for Disinfection or as a Disinfectant

A lysozyme of the present invention may be useful as a disinfectant orused for disinfection, e.g., for the treatment of infections in the eyeor the mouth, or for cleaning and disinfection of contact lenses, andfor preventing or removing biofilm on a surface according to U.S. Pat.No. 6,777,223.

A lysozyme of the present invention may also be used in oral care. Forexample, lysozyme can be used alone or in combination with other enzymesor even antimicrobial peptides in toothpaste or other oral careproducts. The polypeptides may be introduced into the oral cavity orapplied to an article that is to be introduced into the oral cavity. Seefor example WO 2008/124764.

In general it is contemplated that the polypeptides of the presentinvention are useful for cleaning, disinfecting or inhibiting microbialgrowth on any surface. Examples of surfaces, which may advantageously becontacted with the polypeptides of the invention are surfaces of processequipment used, e.g., dairies, chemical or pharmaceutical processplants, water sanitation systems, oil processing plants, paper pulpprocessing plants, water treatment plants, and cooling towers. Thepolypeptides of the invention should be used in an amount, which iseffective for cleaning, disinfecting or inhibiting microbial growth onthe surface in question.

The polypeptides of the invention may additionally be used for cleaningsurfaces and cooking utensils in food processing plants and in any areain which food is prepared or served such as hospitals, nursing homes andrestaurants.

Uses of Lysozymes of the Invention in Food Applications

A lysozyme of the present invention may also be used to selectivelyinhibit the uncontrolled growth of Clostridium tyrobutyricum during thematuration of cheeses, in particular, those made from pressed and cookedcurds, e.g., Swiss Cheese, Parmesan, Edam, Gouda, Cheddar, and manyothers.

A lysozyme of the present invention may also be used in wine making, tocontrol or inhibit microbial contamination.

Uses of Lysozymes of the Invention as Treatments

A lysozyme of the present invention may also be used in topicaltreatment of dystrophic and inflammatory lesions of the skin and softtissues. See, e.g., Palmieri and Boraldi, 1977, Arch. Sci. Med. (Torino)134:481-485.

A lysozyme of the present invention may also be used in skin care. Forexample, the polypeptide is applied to the skin of a patient sufferingfrom a skin infection, such as acne. The lysozyme may also be used in awound dressing, which is applied to wounded skin, for example, to aid inhealing of the wound. See, for example, U.S. Application No.2008/0254079.

A lysozyme of the present invention may also be used in lipstick, lipbalm, lip gel, or lip gloss. For example, such products can be used fortreatment of a localized lip infection, for example, a cold sore. See,for example, U.S. Application No. 2008/0254079.

A lysozyme of the present invention may also be used in the treatment ofbronchopulmonary diseases.

A lysozyme of the present invention may also be used as digestiveenzymes or digestive aids. A lysozyme of the present invention may alsobe used to improve the use of dead/live bacteria as a food source, e.g.,by controlling undesirable microbial contaminants.

A lysozyme of the present invention may also be used as a therapeutic ina human or other animal, e.g., to control or inhibit bacterialovergrowth in the intestines of a human suffering from a disease, e.g.,pancreatic disease or an immuno compromised patient.

Uses of Lysozymes of the Invention for Extracting Bacterial Genomic DNA

A lysozyme of the present invention may also be used to aid in theextraction of bacterial genomic DNA. In order to be able to sequencebacterial DNA, the bacterial cell wall needs to be broken down toisolate the DNA inside it. The cell wall of especially gram positivebacteria can be difficult to break down.

Hen egg white lysozyme is the standard enzyme used for DNA isolationfrom gram positive bacteria and works by hydrolyzing the peptidoglycanchains present in the cell wall thereby aiding in the degradation of thecell walls. However some gram positive cell walls are not degraded byhen egg white lysozymes. For example, it is recommended that cells from,e.g., Staphylococcus aureus are lysed with lysostaphin as described byPitcher and Saunders, 1989, App. Environ. Microbiol. 56(3): 782-787.However, these methods do not work for all types of gram positivebacteria and novel lysozymes thus potentially offer access to novelgenomes that cannot be isolated with commercial lysozyme solutions.

Other Uses of Lysozymes of the Invention

A lysozyme of the present invention may also be used to controlmicrobial growth in a fermentation process, such as, in making ethanolor other products from biomass. See, e.g., WO 2007/109750. Accordingly,the lysozyme may be used, e.g., in a process for producing afermentation product comprising (a) liquefying and/or saccharifying acarbohydrate material and (b) fermenting using a fermentation organism,wherein a lysozyme of the present invention is applied to thefermentation process before, during and/or after fermentationconcentrations sufficient to kill and/or inhibit growth of bacterialcells.

A lysozyme of the present invention may also be used in controllingmicrobial growth in a fish or shrimp farm.

Other uses include preservation of foods, beverages, cosmetics such aslotions, creams, gels, ointments, soaps, shampoos, conditioners,antiperspirants, deodorants, enzyme formulations, or food ingredients.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention having antimicrobialand/or lysozyme activity.

The composition may comprise a polypeptide of the invention as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the composition may comprise multiple enzymatic activities, such as anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the polypeptide composition may be in the form of a granulateor a microgranulate. The polypeptide to be included in the compositionmay be stabilized in accordance with methods known in the art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Animal Feed Compositions

The present invention is also directed to methods for using thepolypeptides of the present invention having lysozyme activity in animalfeed, as well as to feed compositions and feed additives comprising thelysozymes of the invention.

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goats, and cattle, e.g., beef cattleand cows. In a particular embodiment, the animal is a non-ruminantanimal. Non-ruminant animals include mono-gastric animals, e.g., pigs orswine (including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys, geese, ducks and chicken (including but notlimited to broiler chicks, layers); horses (including but not limited tohotbloods, coldbloods and warm bloods), young calves; and fish(including but not limited to salmon, trout, tilapia, catfish and carps;and crustaceans (including but not limited to shrimps and prawns).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal. In the use according to the invention the lysozyme can be fed tothe animal before, after, or simultaneously with the diet. The latter ispreferred. Such lysozyme compositions may of course be mixed with otherenzymes.

The lysozyme can be added to the feed in any form, be it as a relativelypure lysozyme or in admixture with other components intended foraddition to animal feed, i.e., in the form of animal feed additives,such as the so-called pre-mixes for animal feed. In a further aspect,the present invention relates to compositions for use in animal feed,such as animal feed, and animal feed additives, e.g., premixes.

Apart from the lysozyme of the invention, the animal feed additives ofthe invention contain at least one fat-soluble vitamin, and/or at leastone water soluble vitamin, and/or at least one trace mineral, and/or atleast one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.,carotenoids such as beta-carotene, astaxanthin, and lutein; stabilisers;growth improving additives and aroma compounds/flavorings, e.g.,creosol, anethol, deca-, undeca- and/or dodeca-lactones, ionones, irone,gingerol, piperidine, propylidene phthalide, butylidene phatalide,capsaicin and/or tannin; polyunsaturated fatty acids (PUFAs); reactiveoxygen generating species; also, a support may be used that may contain,for example, 40-50% by weight of wood fibres, 8-10% by weight ofstearine, 4-5% by weight of curcuma powder, 4-58% by weight of rosemarypowder, 22-28% by weight of limestone, 1-3% by weight of a gum, such asgum arabic, 5-50% by weight of sugar and/or starch and 5-15% by weightof water.

A feed or a feed additive of the invention may also comprise at leastone other enzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase(EC 3.2.1.22); protease (EC 3.4), phospholipase A1 (EC 3.1.1.32);phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5);phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase suchas, for example, alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC3.2.1.4 or EC 3.2.1.6).

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

Usally fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a protease of the invention, is ananimal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or animal feed at levels of 0.1 ppm to 1000 ppm, preferably0.5 ppm to 200 ppm and more preferably 1 ppm to 100 ppm. Theaforementioned dosing levels can also be used for premixes.

The animal feed composition of the invention may contain at least onevegetable protein, such as that derived from or originating from avegetable, including modified proteins and protein-derivatives.Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal,Alternatively, the vegetable protein source is material from one or moreplants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach orquinoa. Other examples of vegetable protein sources are rapeseed,sunflower seed, cotton seed, and cabbage and cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Destillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can, e.g., be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, for mashfeed a solid or liquid enzyme formulation may be added before or duringthe ingredient mixing step. For pelleted feed the (liquid or solid)lysozyme/enzyme preparation may also be added before or during the feedingredient step. Typically a liquid lysozyme/enzyme preparation is addedafter the pelleting step. The enzyme may also be incorporated in a feedadditive or premix.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of0.5-25 mg enzyme protein per kg animal diet.

Cleaning or Detergent Compositions

The lysozyme of the invention may be added to and thus become acomponent of a detergent composition, particularly in a liquid detergenthaving a pH of 7 or lower.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pretreatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the lysozyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

In one embodiment, the invention is directed to cleaning or detergentcompositions comprising of an enzyme of the present invention incombination with one or more additional cleaning components. The choiceof additional cleaning components is within the skill of the artisan andincludes conventional ingredients, including the exemplary non-limitingcomponents set forth below.

The choice of components may include, for textile care, theconsideration of the type of textile to be cleaned, the type and/ordegree of soiling, the temperature at which cleaning is to take place,and the formulation of the detergent product. Although the componentsmentioned below are categorized according to a particular function, thisshould not be construed as a limitation since the component may have oneor more additional functionalities which the skilled artisan willappreciate.

The cleaning or detergent composition may be suitable for the laundringof textiles such as, e.g., fabrics, cloths or linen, or for cleaninghard surfaces such as, e.g., floors, tables, or dish wash.

The invention also relates to polynucleotides encoding the polypeptides,nucleic acid constructs, vectors, and host cells comprising thepolynucleotides as well as methods of producing and using thepolypeptides.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized.

When included therein the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylethanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternaryammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, andcombinations thereof.

When included therein the detergent will usually contain from about 0.2%to about 40% by weight of a non-ionic surfactant, for example from about0.5% to about 30%, in particular from about 1% to about 20%, from about3% to about 10%, such as from about 3% to about 5%, or from about 8% toabout 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N—(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein the detergent will usually contain from about 0%to about 10% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however, the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see, e.g., review by Hodgdonand Kaler, 2007, Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate(STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers,sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodiumethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 45% of a detergent builder or co-builder, or a mixturethereof. In a dish wash deteregent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry detergents may be utilized. Non-limiting examples of buildersinclude zeolites, diphosphates (pyrophosphates), triphosphates such assodium triphosphate (STP or STPP), carbonates such as sodium carbonate,soluble silicates such as sodium metasilicate, layered silicates (e.g.,SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA),diethanolamine (DEA, also known as iminodiethanol), triethanolamine(TEA, also known as 2,2′,2″-nitrilotriethanol), and carboxymethyl inulin(CMI), and combinations thereof.

The detergent composition may also contain 0-20% by weight, such asabout 5% to about 10%, of a detergent co-builder, or a mixture thereof.The detergent composition may include include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid(HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis(methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid(SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N,N-diaceticacid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N,N′,N′-triacetate (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 2009/102854, U.S. Pat. No.5,977,053.

Bleaching Systems

The detergent may contain 0-50% by weight, such as about 0.1% to about25%, of a bleaching system. Any bleaching system known in the art foruse in laundry detergents may be utilized. Suitable bleaching systemcomponents include bleaching catalysts, photobleaches, bleachactivators, sources of hydrogen peroxide such as sodium percarbonate andsodium perborates, preformed peracids and mixtures thereof. Suitablepreformed peracids include, but are not limited to, peroxycarboxylicacids and salts, percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone®, and mixturesthereof. Non-limiting examples of bleaching systems includeperoxide-based bleaching systems, which may comprise, for example, aninorganic salt, including alkali metal salts such as sodium salts ofperborate (usually mono- or tetra-hydrate), percarbonate, persulfate,perphosphate, persilicate salts, in combination with a peracid-formingbleach activator. The term bleach activator is meant herein as acompound which reacts with peroxygen bleach like hydrogen peroxide toform a peracid. The peracid thus formed constitutes the activatedbleach. Suitable bleach activators to be used herein include thosebelonging to the class of esters amides, imides or anhydrides. Suitableexamples are tetracetylethylene diamine (TAED), sodium4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxydodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)—benzenesulfonate (NOBS), and/or those disclosed in WO98/17767. A particular family of bleach activators of interest wasdisclosed in EP 624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that it is environmental friendly as iteventually degrades into citric acid and alcohol. Furthermore, acetyltriethyl citrate and triacetin has a good hydrolytical stability in theproduct upon storage and it is an efficient bleach activator. Finally,ATC provides a good building capacity to the laundry additive.Alternatively, the bleaching system may comprise peroxyacids of, forexample, the amide, imide, or sulfone type. The bleaching system mayalso comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).The bleaching system may also include a bleach catalyst. In someembodiments, the bleach component may be an organic catalyst selectedfrom the group consisting of organic catalysts having the followingformulae:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g.,in WO 2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242.Suitable photobleaches may for example be sulfonated zincphthalocyanine.

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of poly(ethyleneterephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions of the present invention may also includefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions andthus altering the tint of said fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight. In contrast, fabric hueing agents alter the tint of a surface asthey absorb at least a portion of the visible light spectrum. Suitablefabric hueing agents include dyes and dye-clay conjugates, and may alsoinclude pigments. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group consisting of dyes falling into the Colour Index(C.I.) classifications of Direct Blue, Direct Red, Direct Violet, AcidBlue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO 2005/003274, WO2005/003275, WO 2005/003276 and EP 1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueingagent. The composition may comprise from 0.0001 wt % to 0.2 wt. % fabrichueing agent, this may be especially preferred when the composition isin the form of a unit dose pouch. Suitable hueing agents are alsodisclosed in, e.g., WO 2007/087257 and WO 2007/087243.

Additional Enzymes

In one aspect, the present invention provides a detergent additivecomprising a lysozyme of the present invention. The detergent additiveas well as the detergent composition may comprise one or more[additional] enzymes such as a protease, lipase, cutinase, an amylase,carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase,xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583. Examples ofuseful proteases are the variants described in WO 92/19729, WO 98/20115,WO 98/20116, and WO 98/34946, especially the variants with substitutionsin one or more of the following positions: 27, 36, 57, 76, 87, 97, 101,104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™ Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™ Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases and Cutinases: Suitable lipases and cutinases include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutant enzymes are included. Examples include lipase from Thermomyces,e.g., from T. lanuginosus (previously named Humicola lanuginosa) asdescribed in EP 258068 and EP 305216, cutinase from Humicola, e.g., H.insolens (WO 96/13580), lipase from strains of Pseudomonas (some ofthese now renamed to Burkholderia), e.g., P. alcaligenes or P.pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strainSD705 (WO 95/06720 & WO 96/27002), P. wisconsinensis (WO 96/12012),GDSL-type Streptomyces lipases (WO 2010/065455), cutinase fromMagnaporthe grisea (WO 2010/107560), cutinase from Pseudomonas mendocina(U.S. Pat. No. 5,389,536), lipase from Thermobifida fusca (WO2011/084412), Geobacillus stearothermophilus lipase (WO 2011/084417),lipase from Bacillus subtilis (WO 2011/084599), and lipase fromStreptomyces griseus (WO 2011/150157) and S. pristinaespiralis (WO2012/137147).

Other examples are lipase variants such as those described in EP 407225,WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO00/34450, WO 00/60063, WO 01/92502, WO 2007/87508 and WO 2009/109500.

Preferred commercial lipase products include include Lipolase™, Lipex™;Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally fromGenencor) and Lipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g., acyltransferases with homologyto Candida antarctica lipase A (WO 2010/111143), acyltransferase fromMycobacterium smegmatis (WO 2005/56782), perhydrolases from the CE 7family (WO 2009/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).

Amylases: Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g., a special strain of Bacillus licheniformis, described inmore detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™ Natalase™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™(from Genencor International Inc.).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants:

The detergent compositions of the present invention can also containdispersants. In particular powdered detergents may comprise dispersants.Suitable water-soluble organic materials include the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents:

The detergent compositions of the present invention may also include oneor more dye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

Fluorescent Whitening Agent:

The detergent compositions of the present invention will preferably alsocontain additional components that may tint articles being cleaned, suchas fluorescent whitening agent or optical brighteners. Where present thebrightener is preferably at a level of about 0.01% to about 0.5%. Anyfluorescent whitening agent suitable for use in a laundry detergentcomposition may be used in the composition of the present invention. Themost commonly used fluorescent whitening agents are those belonging tothe classes of diaminostilbene-sulphonic acid derivatives,diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.Examples of the diaminostilbene-sulphonic acid derivative type offluorescent whitening agents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N−2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)—1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the invention include the 1-3-diaryl pyrazolines andthe 7-alkylaminocoumarins. Suitable fluorescent brightener levelsinclude lower levels of from about 0.01, from 0.05, from about 0.1 oreven from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.

Soil Release Polymers:

The detergent compositions of the present invention may also include oneor more soil release polymers which aid the removal of soils fromfabrics such as cotton and polyester based fabrics, in particular theremoval of hydrophobic soils from polyester based fabrics. The soilrelease polymers may for example be nonionic or anionic terephthaltebased polymers, polyvinyl caprolactam and related copolymers, vinylgraft copolymers, polyester polyamides see for example Chapter 7 inPowdered Detergents, Surfactant science series volume 71, Marcel Dekker,Inc. Another type of soil release polymers are amphiphilic alkoxylatedgrease cleaning polymers comprising a core structure and a plurality ofalkoxylate groups attached to that core structure. The core structuremay comprise a polyalkylenimine structure or a polyalkanolaminestructure as described in detail in WO 2009/087523 (hereby incorporatedby reference). Furthermore, random graft co-polymers are suitable soilrelease polymers Suitable graft co-polymers are described in more detailin WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose deriviatives such as those described in EP1867808 or WO 2003/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof. Suitable cellulosic polymersinclude methyl cellulose, carboxy methyl cellulose, ethyl cellulose,hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, estercarboxy methyl cellulose, and mixtures thereof.

Anti-Redeposition Agents:

The detergent compositions of the present invention may also include oneor more anti-redeposition agents such as carboxymethylcellulose (CMC),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethyleneand/or polyethyleneglycol (PEG), homopolymers of acrylic acid,copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Biofilms

Microorganisms growing in biofilms are less susceptible to all types ofantimicrobial agents than the same microorganisms when grown inconventional suspension cultures.

It is well known that starved bacteria can be much less susceptible to avariety of antimicrobial challenges. For example, a number of classicalantibiotics such as penicillin, perform poorly in slow or non dividingbacteria. Because lysozyme attacks and destroys the peptidoglycan layerregardless of the growth state of the bacteria, it remains effective.

Biofilm Control; Example Dental Water Lines:

Biofilm buildup within a dental water line can contain biofilmsconsisting of Pseudomonas aeruginosa, Proteus mirabilis, Legionella sp.to name but a few. There is also the possibility of colonisation ofspecies generally found within the oral cavity as a result of thefailure of anti retraction valves within the system. The risk of crossinfection becomes even more of a potential risk of course whenimmuno—compromised patients are involved and in this day and age thenumbers of patients within this category continues to steadily increase.The need exists for effective control of bacterial biofilm accumulationin dental water lines. A review of biofilms can be found: Watnick andKolter, 2000, “Biofilm, city of microbes”, J. Bacteriol.182(10):2675-2679.

A typical example of a commercial throat lozenge product is Lysopaineproduced by: Boehringer Ingelheim (France)

Active ingredients:

BACITRACIN 200 U.I.

(to 65 iu/mg)

PAPAIN 2 mg

to 30 NK/mg

LYSOZYME CHLORHYDRATE 5 mg

to 26000 U FIP/mg: units determined by measuring OD kinetics of lysis ofbacteria suspended in buffer. The unit determination was measured bylysis induced change in turbidity of a bacterial culture suspended inbuffer.

Non active ingredients:

SACCHARIN excipient

MAGNESIUM STEARATE excipient

Menthol aromatisant

SORBITOL excipient

For local treatment of point infections limited to the buccal membranesof the oropharynx. Caution, if clinical indications of a generalbacterial infection are evident, antibiotic therapy is advised.

Toothpaste:

Lysozyme can be used alone or in combination with other enzymes or evenantimicrobial peptides. Examples of other enzymes are glucose oxidaseand lactoperoxidase.

A typical toothpaste composition including lysozyme is “Biotene” byLaclede, Inc., 2030 East University Drive, Rancho Domiguez, Calif.90220, USA.

Active Ingredients

Contains: Lactoperoxidase (100 gm)

Inactive Ingredients

Glucose Oxidase, Lysozyme, Sodium Monofluorophosphate, Sorbitol,Glycerin, Calcium Pyrophosphate, Hydrated Silica, Zylitol, CelluloseGum, Flavor, Sodium Benzoate, Beta-d-glucose, Potassium Thiocyanate

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

Aspergillus aculeatus CBS 172.66 was used as the source of DNA forobtaining the coding region encoding the GH23 lysozyme candidate.According to Central Bureau vor Schnimmelkulture, Aspergillus aculeatusCBS 172.66 was isolated by K. B. Raper in 1962 from tropical soil.

Aspergillus oryzae MT3568 strain was used for expression of the A.aculeatus gene encoding the enzyme. A. oryzae MT3568 is an amdS(acetamidase) disrupted gene derivative of Aspergillus oryzae JaL355 (WO02/40694) in which pyrG auxotrophy was restored by disrupting the A.oryzae acetamidase (amdS) gene with the pyrG gene. According to CentralBureau vor Schnimmelkulture, Acremonium alkalophilum CBS 114.92 wasisolated by A. Yoneda in 1984 from the sludge of pig faeces compost nearTsukui Lake, Japan.

Media and Solutions

YP medium was composed of 10 g of yeast extract, 20 g of Bactopeptone,and deionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

Horikoshi agar medium was composed of: 1% (w/v) Dextrose, 1% solublestarch, 0.5% (w/v) peptone, 0.5% (w/v) yeast extract, 0.02% (w/v)MgSO₄.7H₂O, 0.1% (w/v) K₂HPO₄, and 15 g (w/v) of Bacto-agar. 1% (w/v)Na₂CO₃ was added separately after sterilization.

PDA agar plates were composed of potato infusion (potato infusion wasmade by boiling 300 g of sliced (washed but unpeeled) potatoes in waterfor 30 minutes and then decanting or straining the broth throughcheesecloth. Distilled water was then added until the total volume ofthe suspension was one liter, followed by 20 g (w/v) of dextrose and 20g (w/v) of agar powder. The medium was sterilized by autoclaving at 15psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition,Revision A, 1998).

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salt solution, and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCl and TritonX-100 (50 μl/500 ml) were added.

LB agar plates were composed of 37 g of LB agar and deionized water to 1liter.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 ml of COVE trace metal solution, and deionized water to 1liter.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Dap-4C medium was composed of 20 g of dextrose, 10 g of maltose, 11 g ofMgSO₄.7H₂O, 1 g of KH₂PO₄, 2 g of citric acid, 5.2 g of K₃PO₄.H₂O, 0.5 gof yeast extract (Difco), 1 ml of antifoam, 0.5 ml KU6 trace metalssolution, 2.5 g of CaCO₃, and deionized water to 1 liter. The medium wassterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998). Before use, 3.5 ml ofsterile 50% (NH₄)₂HPO₄ and 5 ml of sterile 20% lactic acid were addedper 150 ml of medium.

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone and 2%glucose.

Example 1: Lysozyme Assay

Xanthomonas campestris is the production organism for all xanthan gumproduction. The separation of Xanthomonas cells from the highly viscousxanthan solution is a cost-intensive process in the industrialproduction (Homma et al., EP 690072, Murofushi et al., EP 718311, U.S.Pat. No. 5,702,927). Nowadays, the favoured method for recoveringxanthan from the fermentation liquid is precipitation with alcohol,mainly isopropanol, after pasteurization to destroy bacterial cells andenzymes (Cottrell et al., 1978, “Xanthan gum: A unique bacterialpolysaccharide for food applications”, Ind. Microbiol, 19:177).Subsequently the xanthan/cell debris precipitate is spray dried andmilled to a powder. The alcohol is recovered by distillation. Because ofthe significant amounts of Xanthomonas cell wall debris in somecommercial preparations of xanthan gum, and this debris is peptidoglycanrich Xanthomonas cell wall material, the gum can be used as a convenientassay for peptidoglycan degrading activity.

Solid Plate Assay:

Commercially prepared Xanthan gum (Sigma # G−1253) is dissolved in abuffered solution or bacterial growth media to 0.5% w/v in the presenceof 0.7% agarose and then autoclaved. Enzyme preparations, supernatantsor whole organisms are either deposited in wells cut out of the Bactoagar plates or deposited directly on the surface of the media. Thepreparations are able to form clearing zones in the plates. Theseclearing zones can indicate degradation of bacterial cell wall material.

Liquid Clearing Assay:

Commercially prepared xanthan gum is dissolved in a buffered solution inthe presence or absence of sodium chloride. The solution is autoclavedand used for studies of xanthan gum clearing. Enzyme preparations,supernatants or whole organisms are added to the assay medium andincubated. Resulting treatments are measured in a spectrophotometer todetermine the OD of the solution. Typically, a wavelength of 600 nm isused.

Example 2: Cloning and Characterization of the Aspergillus aculeatusGH23 Lysozyme (SEQ ID NO: 2)

Genomic sequence information was generated by the U.S. Department ofEnergy Joint Genome Institute (JGI). According to Central Bureau vorSchnimmelkulture, Aspergillus aculeatus CBS 172.66 was isolated by K. B.Raper in 1962 from tropical soil. A preliminary assembly of the genomewas downloaded from JGI and analyzed using the Pedant-Pro™ SequenceAnalysis Suite (Biomax Informatics AG, Martinsried, Germany). Genemodels constructed by the software were used as a starting point fordetecting GH23 homologues in the genome. More precise gene models wereconstructed manually using multiple known GH23 protein sequences as aguide.

To generate genomic DNA for PCR amplification, Aspergillus aculeatus CBS172.66 was propagated on PDA agar plates by growing at 26° C. for 7days. Spores harvested from the PDA plates were used to inoculate 25 mlof YP+2% glucose medium in a baffled shake flask and incubated at 26° C.for 48 hours with agitation at 200 rpm.

Genomic DNA was isolated according to a modified FastDNA® SPIN protocol(Qbiogene, Inc., Carlsbad, Calif., USA). Briefly a FastDNA® SPIN Kit forSoil (Qbiogene, Inc., Carlsbad, Calif., USA) was used in a FastPrep® 24Homogenization System (MP Biosciences, Santa Ana, Calif., USA). Two mlof fungal material from the above cultures were harvested bycentrifugation at 14,000×g for 2 minutes. The supernatant was removedand the pellet resuspended in 500 μl of deionized water. The suspensionwas transferred to a Lysing Matrix E FastPrep® tube (Qbiogene, Inc.,Carlsbad, Calif., USA) and 790 μl of sodium phosphate buffer and 100 μlof MT buffer from the FastDNA® SPIN Kit were added to the tube. Thesample was then secured in the FastPrep® Instrument (Qbiogene, Inc.,Carlsbad, Calif., USA) and processed for 60 seconds at a speed of 5.5m/sec. The sample was then centrifuged at 14000×g for two minutes andthe supernatant transferred to a clean EPPENDORF® tube. A 250 μl volumeof PPS reagent from the FastDNA® SPIN Kit was added and then the samplewas mixed gently by inversion. The sample was again centrifuged at14000×g for 5 minutes. The supernatant was transferred to a 15 ml tubefollowed by 1 ml of Binding Matrix suspension from the FastDNA® SPIN Kitand then mixed by inversion for two minutes. The sample was placed in astationary tube rack and the silica matrix was allowed to settle for 3minutes. A 500 μl volume of the supernatant was removed and discardedand then the remaining sample was resuspended in the matrix. The samplewas then transferred to a SPIN filter tube from the FastDNA® SPIN Kitand centrifuged at 14000×g for 1 minute. The catch tube was emptied andthe remaining matrix suspension added to the SPIN filter tube. Thesample was again centrifuged (14000×g, 1 minute). A 500 μl volume ofSEWS-M solution from the FastDNA® SPIN Kit was added to the SPIN filtertube and the sample was centrifuged at the same speed for 1 minute. Thecatch tube was emptied and the SPIN filter replaced in the catch tube.The unit was centrifuged at 14000×g for 2 minutes to “dry” the matrix ofresidual SEWS-M wash solution. The SPIN filter was placed in a freshcatch tube and allowed to air dry for 5 minutes at room temperature. Thematrix was gently resuspended in 100 μl of DES (DNase/Pyrogen freewater) with a pipette tip. The unit was centrifuged (14000×g, 1 minute)to elute the genomic DNA followed by elution with 100 μl of 10 mM Tris,0.1 mM EDTA, pH 8.0 by renewed centrifugation at 14000×g for 1 minuteand the eluates were combined. The concentration of the DNA harvestedfrom the catch tube was measured by a UV spectrophotometer at 260 nm.

Construction of an Asperqillus oryzae Expression Vector ContainingAsperqillus aculeatus CBS 172.66 Genomic Sequence Encoding a Family GH23Polypeptide P24DZF Having Lysozyme Activity.

Two synthetic oligonucleotide primers shown in table 1 below weredesigned to PCR amplify the Aspergillus aculeatus CBS 172.66 P8EH GH23gene from the genomic DNA. An IN-FUSION™ Cloning Kit (Clontech, MountainView, Calif., USA) was used to clone the fragment directly into theexpression vector pDau109 (WO 2005/042735).

TABLE 1 Primers used for GH23 PCR Amplification GH23 Specific Specificgene forward primer reverse primer Aspergillus F-P8EH R-P8EH aculeatus5′-ACACAACTGGGG 5′-AGATCTCGAGAA CBS 172.66 ATCCACCATGCAGTTGCTTACTATGCGCTC GAACAACTTCCTTC AGGGTGCACT-3′ T-3′ (SEQ ID NO: 10)(SEQ ID NO: 9)

Bold letters represent coding sequence. The underlined sequence ishomologous to the insertion sites of pDau109.

The PCR reaction (25 μl) was composed of 12.5 μl of 2×IPROOF™ HF MasterMix, 0.5 μl of primer F-P24DZF (100 μM), 0.5 μl of primer R-P24DZF (100μM), 0.5 μl of genomic (100 ng/μl), and 11 μl of deionized water. ThePCR reaction was incubated in a DYAD® Dual-Block Thermal Cycler (MJResearch Inc., Waltham, Mass., USA) programmed for 1 cycle at 98° C. for30 seconds; 30 cycles each at 98° C. for 10 seconds, 55° C. for 10seconds, and 72° C. for 60 seconds; and 1 cycle at 72° C. for 10minutes. Samples were cooled to 10° C. before removal and furtherprocessing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where an approximately 770 bp productband was observed. The remaining PCR reaction was purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The fragment was then cloned into Hind III and Bam HI digested pDau109using an IN-FUSION™ Cloning Kit resulting in plasmid pP8EH. Cloning ofthe P24DZF gene into Hind III-Bam HI digested pDau109 resulted in thetranscription of the Aspergillus aculeatus P24DZF gene under the controlof a NA2-tpi double promoter. NA2-tpi is a modified promoter from thegene encoding the Aspergillus niger neutral alpha-amylase in which theuntranslated leader has been replaced by an untranslated leader from thegene encoding the Aspergillus nidulans triose phosphate isomerase.

The cloning protocol was performed according to the IN-FUSION™ CloningKit instructions generating a P24DZF GH23 construct. The treated plasmidand insert were transformed into Fusion Blue™ E. coli cells (Clontech,Mountain View, Calif., USA) according to the manufacturer's protocol andplated onto LB plates supplemented with 50 μg of ampicillin per ml.After incubating at 37° C. overnight, colonies were seen growing underselection on the LB ampicillin plates. Ten colonies transformed with theP24DZF GH23 construct were cultivated in LB medium supplemented with 50μg of ampicillin per ml and plasmid was isolated using a FASTPlasmidmini kit from 5Prime (5 PRIME GmbH, Königstrasse 4a, 22767 Hamburg,Germany) according to the manufacturer's instructions.

Isolated plasmids were sequenced with vector primers and in order todetermine a representative plasmid expression clone that was free of PCRerrors.

Characterization of the Asperqillus Aculeatus CBS 172.66 GenomicSequences Encoding GH23 Polypeptide

DNA sequencing of the Aspergillus aculeatus CBS172.66 P24DZF GH23genomic clone was performed with an Applied Biosystems Model 3730xlAutomated DNA Sequencer using version 3.1 BIG-DYE™ terminator chemistry(Applied Biosystems, Inc., Foster City, Calif., USA) and primer walkingstrategy. Nucleotide sequence data were scrutinized for quality and allsequences were compared to each other with assistance of PHRED/PHRAPsoftware (University of Washington, Seattle, Wash., USA). The sequenceobtained was identical to the sequence from the JGI and is shown in SEQID NO: 1.

The nucleotide sequence and deduced amino acid sequence of theAspergillus aculeatus P24DZF GH23 gene are shown in SEQ ID NO: 1 and SEQID NO: 2, respectively. The coding sequence is 862 bp including the stopcodon and is interrupted by a single intron of 67 bp (nucleotides 572 to638). The encoded predicted protein is 264 amino acids and is shown inSEQ ID NO: 2. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 245 amino acids.

The Aspergillus oryzae strain MT3568 was used for all experiments.Aspergillus oryzae MT3568 is an amdS (acetamidase) disrupted derivativeof A. oryzae JaL355 (WO 02/40694) in which pyrG auxotrophy was restoredin the process of knocking out the A. oryzae amdS gene. A. oryzae MT3568protoplasts were prepared according to the method of European Patent, EP0238023, pages 14-15. Fresh protoplasts of A. oryzae MT3568 wereprepared and transformed with the P24DZF GH23 plasmid. Plasmid DNA fromthe above mini prep procedure was used to transform A. oryzae MT3568.

Six ul containing about 3.0 μg total DNA was used for thetransformation. The DNA was gently added to 100 μl of A. oryzae MT3568protoplasts and 250 μl of 60% PEG 4000 (Sigma-Aldrich cat. No. 95904).The 60% (W/V) PEG 4000 was prepared in the following manner: PEG 4000powder was dissolved in double distilled H₂O and then heated for 10-20seconds in a microwave oven at 800 watt until dissolved. The dissolvedsolution was cooled down to room temperature and then then adjusted withCaCl₂ solution and Tris-HCl solution (pH 7.5) for a final concentrationof 10 mM of each. After adding the 60% PEG 4000 solution, the tube wasgently mixed and incubated at 37° C. for 30 minutes. The mix was addedto 6 ml of top agar with 10 mM acetamide and plated onto COVE-sorbitolplates with 10 mM acetamide.

The plates were incubated at 37° C. for 3 or more days and then moved to26° C. for two days. Spores from 8 individual colonies were picked byfirst dipping a white 10 μl inoculation pin (Nunc A/S, Denmark) in a0.1% TWEEN® 80 solution, contacting the sporulating colony on theselection plate, and restreaking with the pin onto fresh COVE sorbitolplates containing 10 mM acetamide. After 5 days at 26° C., spores fromthe restreaked colonies were used to inoculate a 96 well deep dish plate(NUNC, cat. no. 260251, Thermoscientific, USA). The wells of the deepdish plate contained 500 uls of either YP+2% glucose or DAP4C media. Theinoculated plate was sealed with gas permeable tape (89009-656,VWR.com). Plates were incubated stationary at 30 C for 5 days.Expression was verified by analysis of 20 uls of harvested culture fluidon SDS-PAGE using a NUPAGE® 10% Bis-Tris gel (Invitrogen, Carlsbad,Calif., USA) and Coomassie blue staining. One transformant was selectedfor further work and designated A. oryzae EXP03899.

Spores of EXP03899 were inoculated into both YP+2% glucose medium andDAP-4C-1 medium (100 mls in 500 ml Erlenmeyer shake flask with baffles).The cultures were incubated at 26° C. and 150 rpm, 3 days and ifnecessary 4 days. An SDS gel was run as above to test protein amount.

Plate Test for Lysozyme Activity

A Spot assay was performed with Xanthan gum, at pH 5, 7 and 8 asdescribed in the section lysozyme plate assay.

A 1.5% Agarose (Invitrogen cat. 15510-027, electrophoresis grade)solution was prepared in the following buffers:

pH ˜5—in water

pH ˜7—in 0.02M potassium phosphate pH 7

pH ˜8—in 0.02M potassium phosphate pH 8

The agarose was autoclaved for 20 minutes at 121° C. 0.5% Xanthan gum(Sigma G1253) was dissolved in the melted 1.5% agarose and the mixturepoured into petri plates. When the plates were set, sample applicationwells were made with a P-1000 pippette tip (cut off to a 3 mm diameter)attached to a vacuum line.

20 ul of the culture fluid of EXP03899 was deposited in the applicationwells and incubated at 37° C. over night. Samples with lysozyme activitywere observed by clearing zones where the cell debris in the xanthan gumwas observed. Culture fluids from EXP03899 displayed such a clearingzone while the Aspergillus oryzae untransformed transformation hostMT3568 did not produce a noticeable clearing zone. The remaining cultureEXP03899 fluid was filtered though a Fast PES Bottle top filter with a0.22 μm cut-off.and stored in aliquots at −20° C. until further use.

Example 3: Cloning and Characterization of Two Acremonioum alkalophilumGH24 Lysozyme Encoding Genes (SEQ ID NOs: 4 and 6)

Genomic sequence information was generated by the U.S. Department ofEnergy Joint Genome Institute (JGI). According to Central Bureau vorSchnimmelkulture, Acremonium alkalophilum CBS 114.92 was isolated by A.Yoneda in 1984 from the sludge of pig faeces compost near Tsukui Lake,Japan. A preliminary assembly of the genome was downloaded from JGI andanalyzed using the Pedant-Pro™ Sequence Analysis Suite (BiomaxInformatics AG, Martinsried, Germany). Gene models constructed by thesoftware were used as a starting point for detecting GH24 homologues inthe genome. More precise gene models were constructed manually usingmultiple known GH24 protein sequences as a guide.

Acremonium alkalophilum CBS 114.92 was propagated on Horikoshi agar, pH9for 7 days at 30° C. Mycelia was harvested directly from the plate andDNA was isolated according to the FastDNA SPIN Kit for Soil(www.mpbio.com). The DNA was eluted in 100 ul 10 mM TRIS buffer, 0.1 mMEDTA, pH 7, 5 and stored at 4° C. until use.

The pairs of synthetic oligonucleotide primers shown in table 2 belowwere designed to PCR amplify the A. alkalophilum CBS114.92 P242MS GH24gene or P244A7 GH24 gene from the A. alkalophilum genomic DNA describedin example 2 above.

TABLE 2 Primers used for GH24 and GH25 PCR Amplification GH24 SpecificSpecific gene forward primer reverse primer A. alkalophilum F-P242MSR-P242MS CBS 114.92 5′-ACACAACTGGGG 5′-AGATCTCGAGAA GH24 P242MSATCCACCATGGCCAA GCTTACTAAGAACAA GGTCTCTACCCT-3′ GCAGGGAGGGC-3′(SEQ ID NO: 11) (SEQ ID NO: 12) A. alkalophilum F-P244A7 R-P244A7CBS 114.92 5′-ACACAACTGGGG 5′-AGATCTCGAGAAG GH24 P244A7 ATCCACCATGGTCTCCTTACTAAGAGCAAGC TTTCAAGCAGCTC-3′ AGGCAGAGC-3′ (SEQ ID NO: 13)(SEQ ID NO: 14)

Bold letters represent coding sequence. The underlined sequence ishomologous to the insertion sites of pDau109.

DNA sequencing of the Acremonium alkalophilum CBS114.92 GH24 genomicclones were performed with an Applied Biosystems Model 3730xl AutomatedDNA Sequencer using version 3.1 BIG-DYE™ terminator chemistry (AppliedBiosystems, Inc., Foster City, Calif., USA) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The sequences obtainedwere identical to the sequences from the JGI.

P242MS GH24 Gene

The nucleotide sequence and deduced amino acid sequence of theAcremonium alkalophilum GH24 gene are shown in SEQ ID NO: 3 and SEQ IDNO: 4, respectively. The coding sequence is 628 bp including the stopcodon and is interrupted by a single intron of 67 bp (nucleotides 268 to334). The encoded predicted protein is 186 amino acids. Using theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6), asignal peptide of 20 residues was predicted. The predicted matureprotein contains 166 amino acids.

P244A7 GH24 Gene

The nucleotide sequence and deduced amino acid sequence of theAcremonium alkalophilum P244A7 GH24 gene are shown in SEQ ID NO: 5 andSEQ ID NO: 6, respectively. The coding sequence is 782 bp including thestop codon and is interrupted by two introns of 81 bp (nucleotides 134to 214) and 170 bp (nucleotides 346-515). The encoded predicted proteinis 176 amino acids. Using the SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6), a signal peptide of 19 residues waspredicted. The predicted mature protein contains 157 amino acids.

Plasmids for P242MS and P244A7 produced and transformed into Aspergillusorzyzae as in example 2. Transformants with culture fluids that producedrecombinant protein product were identified by SDS-PAGE as in example 2and designated: EXP03865, in the case of P242MS, and EXP03890 in thecase of P244A7. Culture fluids from EXP03865 and EXP03890 fermented inboth YP+2% glucose and DAP4C media were spotted on the xanthan bacterialcell debris plates. It was identified that DAP4C produced the bestexpression of the protein while YP+2% glucose produced the bestexpression for EXP03890 in both SDS-PAGE analysis and spot assayactivity.

Example 4: RDA (Radial Diffusion Assays)

Initially, the antimicrobial activity of the culture supernatants andpurified fractions containing the recombinantly expressed lysozymes wasconfirmed using an RDA's as described previously by Lehrer et al.(Lehrer et al., 1991, “Ultrasensitive assays for endogenousantimicrobial polypeptides”, J. Immunol. Methods 137:167-73), withseveral modifications. Briefly, 30 mL of melted 1/10 Mueller-Hintonbroth (MHB) (Sambrook et al., Molecular Cloning: A Laboratory Manual.Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989) with 1%agarose was cooled to 42° C., supplemented to 5.0×10⁵ cfu/mL with S.carnosus ATCC 51365 or E. coli DSM 682 (ATCC 10536) and was poured intoa single-well omnitray (Nunc) plate. The omnitray plate was overlayedwith a TSP plate (Nunc) and left to solidify. After 1 h, the TSP platewas removed; leaving 96 1-mm wells in which 10 μL of the compound ofinterest could be tested.

10 μl of the test solution is spotted pr. well and the plates areincubated O/N at 37° C. The following day clearing zones indicated nogrowth of test bacteria and thereby antimicrobial activity. The clearingzones were visualized by colouring with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellowtertrazole), that is reduced to purple formazan in living cells(Mosmann, 1983, “Rapid colorimetric assay for cellular growth andsurvival: application to proliferation and cytotoxicity assays”, Journalof Immunological Methods 65 (1-2): 55-63). This colouring provides for adark colouring of living cells and no colouring of the clearing zoneswithout living cells.

The Aspergillus fumigatus GH25 lysozyme (prepared as disclosed inKorczynska et al., 2010, Acta Cryst. F66, 973-977) was included in thetest as a reference. The purified samples shown in table 3 below havebeen tested in the RDA assay.

TABLE 3 Radial Diffusion Assay of GH24 Lysozyme Lysozyme Stock conc.Dilution 0.7 ug/ul Dilution 0.35 ug/ul A. alcalophilum GH24  1.4 ug/ul37.5 ul enz. + 37.5 ul water 18.8 ul enz. + 56.2 ul water SEQ ID NO: 6A. alcalophilum GH25 0.77 ug/ul 68.2 ul enz. + 6.8 ul water  34.1 ulenz. + 40.9 ul water SEQ ID NO: 8 A. fumigatus GH25 12.2 ug/ul  4.3 ulenz. + 70.6 ul water  2.2 ul enz. + 72.8 ul water (reference)Measurement of Clearing Zones

The experiment was performed in triplicate with all resulting in samemeasured clearing zones/zones of inhibition, see FIG. 1. Table 4 belowshows the clearing zones in mm.

TABLE 4 Antimicrobial Clearing Zones of GH24 Lysozyme AgainstStaphylococcus carnosus and Escherichia coli. 0.7 μg/μl 0.35 μg/μl 0.7μg/μl 0.35 μg/μl S. carnosus S. carnosus E. coli E. coli A. alcalophilum12 10 16 14 GH24 SEQ ID NO: 6 A. alcalophilum faint faint 8 (cloudy) 6(cloudy) GH25 SEQ ID NO: 8 A. fumigatus GH25 11 10 10  8 (reference)

The purified lysozyme having SEQ ID NO: 6 showed antimicrobial activityagainst viable cells of the gram positive bacteria Staphylococcuscarnosus and the Gram negative bacteria Escherichia coli.

The antimicrobial activity is not present in culture supernatants fromthe untransformed Aspergillus production host (results not shown).

Large clearing zones with non defined borders were observed surroundingthe application zone for A. alcalophilum GH24 (SEQ ID NO: 6). Theexperiment indicates that the A. alcalophilum lysozyme (SEQ ID NO: 6)and the Aspergillus fumigatus GH25 reference lysozyme have differentactivity and specificity against the two bacteria tested in thisexample.

Example 5: Turbidity Assay

The activity of lysozyme was determined by measuring the decrease (drop)in absorbance/optical density of a solution of resuspended Micrococcuslysodeikticus ATTC No. 4698 (Sigma-Aldrich M3770) or Exiguobacteriumundea (DSM14481) measured in a spectrophotometer at 540 nm.

Preparation of Micrococcus lysodeikticus Substrate

Before use the cells were resuspended in citric acid—phosphate buffer pH6.5 to a concentration of 0.5 mg cells/mL and the optical density (OD)at 540 nm was measured. The cell suspension was then adjusted so thatthe cell concentration equalled an OD540=1.0. The adjusted cellsuspension was then stored cold before use. Resuspended cells were usedwithin 4 hours.

Preparation of Citric Acid-Phosphate Buffer pH 6.5

29 mL 0.1 M citric acid was mixed with 61 mL 0.2 M Na₂HPO₄, and the pHwas adjusted with HCl or NaOH to pH 6.5.

Preparation of Dried Cells of Exiguobacterium undae (the Substrate)

A culture of E. undae (DSM14481) was grown in 100 mL LB medium (Fluka51208, 25 g/L) in a 500 mL shake-flask at 30° C., 250 rpm overnight. Theovernight culture was then centrifuged at 20° C. and 5000 g for 10minutes, and the pellet was washed two times in sterile milliQ water,and resuspended in Milli-Q water. The washed cells were centrifuged for1 minute at 13000 rpm and as much as possible of the supernatant wasdecanted. The washed cells were dried in a vacuum centrifuge for 1 hour.The cell pellet was resuspended in citric acid—phosphate buffer pH 6.5so that the optical density (OD) at 540 nm=1.

Measurement of Lysozyme Antimicrobial Activity in the Turbidity Assay

The lysozyme sample to be measured was diluted to a concentration of100-200 mg enzyme protein/L in citric acid—phosphate buffer pH 6.5, andkept on ice until use. In a 96 well microtiterplate (Nunc) 200 μL of thesubstrate was added to each well, and the plate was incubated at 25° C.or 37° C. for 5 minutes in a VERSAmax microplate reader (MolecularDevices). Following incubation, the absorbance of each well was measuredat 540 nm (start value). To start the activity measurement, 20 μL of thediluted lysozyme sample was added to each substrate (200 μL) and kineticmeasurement of absorbance at 540 nm was initiated for minimum 30 minutesup to 24 hours at 25° C. or 37° C. The measured absorbance at 540 nm wasmonitored for each well and over time a drop in absorbance is seen ifthe lysozyme has lysozyme activity.

The Aspergillus fumigatus GH25 lysozyme (Korczynska et al., 2010, supra)was included in the test as a reference and the results are shown intable 5 below.

TABLE 5 Lysozyme Activity of GH25 Lysozymes against Micrococcuslysodeikticus and Exiguobacterium undea as measured by Optical DensityDrop Exiguobacterium Micrococcus lysodeikticus undae Temperature 37° C.25° C. 37° C. A. alcalophilum GH24 NT − − (SEQ ID NO: 4) A. alcalophilumGH24 +++ + + (SEQ ID NO: 6) A. fumigatus GH25 + +++ +++ (reference) NTmeans not tested − Means no effect + means small effect ++ means mediumeffect +++ means large effect

Example 6: Expression of P242MS GH24 Protein and P244A7 GH24 Protein inAspergillus oryzae

The constructs comprising the relevant lysozyme gene were used toconstruct expression vectors for Aspergillus. The Aspergillus expressionvectors consist of an expression cassette based on the Aspergillus nigerneutral amylase II promoter fused to the Aspergillus nidulans triosephosphate isomerase non translated leader sequence (Pna2/tpi) and theAspergillus niger amyloglycosidase terminator (Tamg). Also present onthe plasmid was the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source. Theexpression plasmids were transformed into Aspergillus as described inexample 3. For each of the constructs 10-20 strains were isolated,purified and cultivated in shake flasks.

Example 7: Purification of P242MS GH24 Protein and P244A7 GH24 Proteinin Aspergillus oryzae

Purification of P242MS GH24 Protein

The fermentation supernatant with the lysozyme was filtered through aFast PES Bottle top filter with a 0.22 μm cut-off. pH was adjusted to7.5 with 0.1 M NaOH and the resulting solution was concentrated (volumereduced by a factor of 8) on a Ultra Filtration Unit (Sartorius) with a5 kDa cut-off membrane.

After pretreatment about 55 ml of the lysozyme containing solution waspurified by chromatography on Q Sepharose, approximately 50 ml in a XK26column, using as buffer A 50 mM TRIS pH 7.5, and as buffer B 50 mMTRIS+1 M NaCl pH 7.5. The fractions from the column were pooled based onthe chromatogram (absorption at 280 and 254 nm) and SDS-PAGE analysis.The pooled fractions were buffer-changed into 50 mM Na-acetate, pH 5.5and concentrated on a Vivacell 250 ml, 5 kDa PES filter.

The molecular weight, as estimated from SDS-PAGE, was approximately 20kDa and the purity was >95%.

Purification of P244A7 GH24 Protein

The fermentation supernatant with the lysozyme was filtered through asandwich of four Whatman glass microfiber filters (2.7, 1.6, 1.2 and 0.7micrometer) and then through a Fast PES bottle top filter with a 0.22 μmcut-off. pH was adjusted to 4.5 with 10% acetic acid. After thepH-adjustment the solution became a little cloudy and this was removedby filtration through a Fast PES Bottle top filter with a 0.22 μmcut-off.

After pretreatment about 970 ml of the lysozyme containing solution waspurified by chromatography on SP Sepharose, approximately 50 ml in aXK26 column, using as buffer A 50 mM Na-acetate pH 4.5, and as buffer B50 mM Na-acetate+1 M NaCl pH 4.5. The fractions from the column werepooled based on the chromatogram (absorption at 280 and 254 nm) andSDS-PAGE analysis. The pooled fractions were buffer-changed into 50 mMNa-acetate, pH 5.5 and concentrated using Amicon spin filters with a 10kDa cut-off.

The molecular weight, as estimated from SDS-PAGE, was approximately 20kDa and the purity was >90%.

The invention claimed is:
 1. A variant polypeptide having lysozymeactivity, which has at least 95% sequence identity and comprises one ormore amino acid substitutions to a sequence selected from the groupconsisting of amino acids 20 to 264 of SEQ ID NO: 2, amino acids 21 to186 of SEQ ID NO: 4, and amino acids 20 to 176 of SEQ ID NO:
 6. 2. Thevariant polypeptide of claim 1, which has at least 95% sequence identityto the sequence of amino acids 20 to 264 of SEQ ID NO:
 2. 3. The variantpolypeptide of claim 1, which has at least 97% sequence identity to thesequence of amino acids 20 to 264 of SEQ ID NO:
 2. 4. The variantpolypeptide of claim 1, which has at least 95% sequence identity to thesequence of amino acids 21 to 186 of SEQ ID NO:
 4. 5. The variantpolypeptide of claim 1, which has at least 97% sequence identity to thesequence of amino acids 21 to 186 of SEQ ID NO:
 4. 6. The variantpolypeptide of claim 1, which has at least 95% sequence identity to thesequence of amino acids 20 to 176 of SEQ ID NO:
 6. 7. The variantpolypeptide of claim 1, which has at least 97% sequence identity to thesequence of amino acids 20 to 176 of SEQ ID NO:
 6. 8. A detergentcomposition comprising the variant polypeptide of claim 1 and asurfactant.
 9. The detergent composition of claim 8, which furthercomprises one or more further enzymes selected from the group comprisingof amylases, catalases, cellulases, cutinases, haloperoxygenases,lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases,xanthanases, and xyloglucanases, or any mixture thereof.
 10. Thedetergent composition of claim 8, further comprising of one or morecomponents selected from the group consisting of builders, hydrotopes,bleaching systems, polymers, fabric hueing agents, adjunct materials,dispersants, dye transfer inhibiting agents, fluorescent whiteningagents, soil release polymers and anti-redeposition agents.
 11. Ananimal feed composition comprising the variant polypeptide of claim 1.12. The animal feed composition of claim 11, which further comprises ofone or more amylases; galactanases; alpha-galactosidases;beta-glucanases, phospholipases, phytases, proteases, and xylanases, orany mixture thereof.
 13. An animal feed additive comprising (a) at leastone variant polypeptide of claim 1; and (b) at least one fat-solublevitamin, at least one water-soluble vitamin, and/or at least one tracemineral.
 14. The animal feed additive of claim 13, which furthercomprises of one or more amylases, galactanases, alpha-galactosidases,beta-glucanases, phospholipases, phytases, proteases, and xylanases, orany mixture thereof.